II.

Tissue processing
3. Exchange ethanol with xylene in the following sequence
Ethanol: Xylene (1:1): 50 min
Xylene : 50 min
Xylene : 50 min

4. Exchange xylene with paraffin at 58 0 C

Xylene: Paraffin (1:1): 60 min
Paraffin: 120 min
Paraffin: 120 min

II I. Tissue embeding
。
cω 一一月呵》

1. Put small amount of molten paraffin in mold , dispensing from paraffin reservoir
2. Transfer tissue into mold by warm forceps , placing cut side down

3. Transfer mold to cold pla悟 ， paraffin will solidify in a thin layer which holds the tissue in position
2 月-σ

4. Add hot paraffin to the mold from the paraffin dispenser. Be sure there is enough paraffin to cover the face of the plastic cassette

5. Paraffin should solidify in 30 minutes

。且 -mwm

IV.Tissue Sectioning
。

1. Placed tissue blocks face down on an ice block or cold plate for 10-30 minutes
cω 一-門呵

2. Place a fresh blade on the microtome. Blades may be used to section up to 10 blocks , but replace if sectioning becomes problematic

3. Insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane
mmmc 一月的

4. Set the dial to cut 10IJm sections to order to plane the block; once it is cutting smoothly , set to 4IJm sections

5. Pick sections and float them on the surface of the cold water. Float the sections onto the surface of clean glass slides

6. Place the slides with paraffin sections on the surface of the 56 0 C water bath to bond the tissue to the glass

V.l mmunohistochemical staining (IHC)

Dewaxing & Rehydrate
1. Place the slides with 4IJm paraffin sections on the warming block in a 60 C oven for 30 minutes
0

2. Perform the following washes

Xylene: twic巴 ， each for 5 min
100% ethanol: twice . each for 5 min
95% ethanol: 5 min
75 % ethanol: 5 min
50 % ethanol: 5 min
Rinse slides with deionized water

Antigen retrieval (Heat-Induced Epitope Retrieval )

1. Fill individual plastic container(s) with appropriate retrieval solution and add enough deionized water to pressure cooker chamber or vegetable steamer
2. Bring slides to a boil in antigen retrieval solution for 15 min in the pressure cooker or 30 min in the vegetable steamer

3. Transfer slides to room temperature Tris-Buffered Saline (TBS)

4. Tissues are now ready for blocking and primary antibody incubation

會 Gen品x International
Tel 886.3.6208988
USA
Toll-free 1.877.GeneTex(1.877 .4 36.3839)
Fax 886.3.6209098 Fax 1.949.309.2888
101010.gelletex.co lJI Address 6F-2 , NO.89 , Dongmei Rd. , Hsinchu 300 , Taiwan (R.O.C.) Address 2456 Alton Parkway , Irvine , CA 92606 USA
E-mail info@genetex.com E-mail info@genetex.com

• Your Expertise, Our Antibodies, Accelerated Di scovery. In!erna!ional

USA
Tel: 886.3.6208988
Tel: 1.949.553.1900 Toll-free: 1.877 .4 36.3839
 IHC staining
1. Wash sections in TBS twice. each for 3 min

2. lf using an HRP conjugate for detection , blocking of endogenous peroxidase can be performed here by incubation of sections in 3% H202 for 10 min

3. Wash section in TBS twice. each for 3 min

4. Block each section with blocking bu行er for 30 min at RT

5. Remove blocking buffer and add primary antibody at proper dilution and incubate for overnight at 40 C

6. Remove antibody solution and wash sections in TBS three times , each for 5 min

7. Add secondary antibody , and incubate for 30 min at RT

8. Prepare avidin-biotin-enzyme complex (ABC) reagent according to the manufacturer's instructions
。

9. Remove secondary antibody solution and wash sections in TBS three times , each for 5 min
cω 一一月呵》

10. Add ABC reagent to each section and incubate for 30 min at RT

11. Remove ABC reagent and wash sections in TBS three times ,each for 5 min

12. Add DAB to each section and monitor staining closely

2 月-σ

13. As soon as the sections develop , immerse slides in ddH20

。且 -mwm

14. Counter-stain sections in hematoxylin

15. Wash sections in ddH20 twice. each for 3 min

。

16. Dehydrate sections

cω 一-門呵

75 % ethanol: 3 min
95% ethanol: 3 min
100% ethanol: 5 min
Xylene: 5 min
mmmc 一月的

17. Mount sections with coverslip

會 Gen品x International
Tel 886.3.6208988
USA
Toll-free 1.877.GeneTex(1 .877 .4 36.3839)
Fax 886.3.6209098 Fax 1.949.309.2888
101010.gelletex.co lJI Address 6F-2 , NO.89 , Dongmei Rd. , Hsinchu 300 , Taiwa n (R.O.C.) Address 2456 Alton Parkway , Irvine , CA 92606 USA
E-mail info@genetex.com E-mail info@genetex.com

• Your Expertise, Our Antibodies, Accelerated Di scovery. I n!erna!ional

USA
Tel: 886.3.6208988
Tel: 1.949.553.1900 Toll-free: 1.877 .4 36.3839
 GeneTex GeneTex started as a
。
cω 一一月呵》

Our corporate mission , to provide our customers with quality reagents and to accelerate life sciences research , reflects the philosophy and approach we

employ when we manufacture our products. Through extensive research , development , and quality-assurance testing , we have produced and validated a

comprehensive collection of antibodies and research reagents

2 月-σ。且 -mwm

Due to GeneTex's experience in antibody production , we are uniquely capable of providing

technical suppo吋 and troubleshooting assistance for our customers

。
cω 一-門呵

吧
mmmc 一月的

C電替步 An心
旬沁
tii旬9
hrm
P昕T甘
「o1
凹
ir
r

Our
Antibodies P仙川川
叫
urr叫i2
芯:: :/ pept ide F L

Y Conjugat 清

會 Gen品x International
Tel 886.3.6208988
USA
Toll-free 1.877.GeneTex(1.877 .4 36.3839)
Fax 886.3.6209098 Fax 1.949.309.2888
101010.gelletex.co lJI Address 6F-2 , No.89 , Dongmei Rd. , Hsinchu 300 , Taiwan (R.O .C.) Address 2456 Alton Parkway , Irvine , CA 92606 USA
E-mail info@genetex.com E-mail info@genetex.com

• Your Expertise, Our Antibodies, Accelerated Di scovery. 1n!erna!ional

USA
Tel: 886.3.6208988
Tel: 1.949.553.1900 Toll-free: 1.877 .4 36.3839
 Selection you need
Eomprehensive C 璽ÐHM

Genetex currently 0仟ers more than 55 ,000 products including over 45 ,000 primary antibodies that cover eleven different research areas. We continue to

build upon our product po吋folio to ensure that novel targets are available to researchers and that Genetex keeps pace with new scientific discovery
。
cω 一一月呵》

In addition to our comprehensive catalog of primary antibodies , GeneTex also 0仟ers a wide variety of quality reagents , kits and services

Angiogenesis
2 月-σ

Apoptosis
。且 -mwm

Blood related

Cancer related
。

CD marker
cω 一-門呵

ζell communication
Cell cycle
mmmc 一月的

Disease related
DNA replication/repair/recombination
Gene expression
GP 仁 R

Growth factor
Housekeeping gene
Immune system
lon transport
Kinase and phosphatase
Membrane transport

Metabolism
Neuroscience
Protein folding and degradation
Signal transduction

Stem cell
-oomwo∞ w
% 、4 。V1~ i'J ~

。 。 OÒ O O o o

會 Gen品x International
Tel 886.3.6208988
USA
Toll-free 1.877.GeneTex(1.877 .4 36.3839)
Fax 886.3.6209098 Fax 1.949.309.2888
101010.gelletex.co lJI Address 6F-2 , No.89 , Dongmei Rd. , Hsinchu 300 , Taiwan (R.O .C.) Address 2456 Alton Parkway , Irvine , CA 92606 USA
E-mail info@genetex.com E-mail info@genetex.com

• Your Expertise, Our Antibodies, Accelerated Di scovery. 1n!erna!ional

USA
Tel: 886.3.6208988
Tel: 1.949.553.1900 Toll-free: 1.877 .4 36.3839